Incubated in hypotonic medium (phosphate buffer saline; 0.45 glucose; 1PLOS One | plosone.orgChromatin Relaxation Triggers p19INK4d Induction(-685/2684). Plasmid pE2WTx4CAT, encoding the chloramphenicol acetyltransferase (CAT) reporter gene driven by an E2 core promoter and four copies on the E2F enhancer [49], was kindly provided by M. Imperiale (University of Michigan Healthcare School). Cells had been transfected following the typical AS2521780 PKC calcium phosphate precipitation approach primarily as previously described [50]. Briefly, cells seeded in 6-well dishes were transfected with 4 mg p19CAT or equal quantity of the mutated version, 5 mg of pCEFLb-galactosidase and expression vectors when indicated. Total DNA amount was adjusted to 15 mg/well with non-specific DNA carrier. Following 16 h, the medium was replaced by serum-free medium, and cells had been additional incubated for 24 h. Cells had been then harvested and CAT and b-galactosidase activities had been determined as previously described [50]. CAT activity was normalized to b-galactosidase activity.Supporting InformationDAD MedChemExpress Figure S1 Cloroquine, TSA and hypotonic medium enhanced MNase accessibility of chromatin. HEK-293 cells were incubated with 100 mM chloroquine (A) or 200 nM TSA (B) or hypotonic medium (50 mM NaCl) (C) as indicated. Right after four h whole nuclei have been isolated and incubated with two U/ml MNase for the indicated occasions. Total genomic DNA was purified and also the pattern of DNA digestion was analyzed by electrophoresis as described in materials and procedures section. Every single figure shows a representative gel of 3 independent experiments with comparable results. Choroquine (Chlo), microccocal nuclease (MNase), hypotonic (Hypo) and isotonic (Iso) medium, markers (M). (TIF) Figure S2 p19 will be the only member of INK4 family that’s induced by chromatin relaxation. HEK-293 cells have been exposed to 100 mM chloroquine, 200 nM TSA or hypotonic medium (50 mM NaCl) for the indicated times. Total RNA (ten mg) extracted from cells at the indicated times had been subjected to northern blot analysis with the 32P-labeled probes specified at the appropriate margin. Figure shows a representative autoradiograph of three independent experiments with equivalent final results. Chloroquine (chlo), hypotonic medium (hypo), b-tubulin (b-tub), neocarzinostatin (NCS). (TIF) Figure S3 Induction of p19 by chromatin modifyingUnscheduled DNA SynthesisNeuro-2a p19AS cells seeded in 6-well dishes were washed with PBS and growth medium was replaced by serum-free medium which was renewed right after 24 h. Inhibition of DNA semiconservative synthesis was confirmed under these conditions. Cells had been treated or not with 50 mM ZnSO4. Soon after 16 h, cells were incubated with 100 mM choroquine and, simultaneously or just after four h, irradiated with 40 J/m2 UV and additional cultured in serum free-medium with ten mCi/ml [3H]thymidine. Ten hours later, cells have been washed three instances with cold PBS, harvested and collected at 3000 g for 5 min. Cells have been lysed with 5 TCA for 30 min and centrifuged at 10,000 g for 10 min. Pellet was washed twice with cold PBS and resuspended in 1 M NaOH. The incorporated radioactivity was quantified by scintillation counting. Unscheduled DNA synthesis was expressed as dpm/mg protein.Cyclobutane Pyrimidine Dimers (CPD) Detection by Immuno-slot Blot AssayThe amount of thymine dimers in the DNA was measured by an immune-slot-blot assay making use of a CPD-specific monoclonal antibody [51]. Roughly 106 Neuro-2a cells have been plated into 60-mm dishes, incubated with 100 mM ch.
